Toll-like receptors (TLRs) are a family of at least ten highly conserved type-I transmembrane proteins (TLR1-TLR10) that are responsible for initiation of innate immune responses in vertebrates. They recognize a variety of pathogen-associated molecular patterns (PAMPs) from bacteria, viruses and fungi and act as a first line of defense against invading pathogens. Engagement of TLRs with their ligands leads to the production of various pro-inflammatory cytokines, chemokines, and effector molecules, depending on the cell type that is activated.
TLRs are characterized by an extracellular amino-terminal leucine-rich repeat (LRR) domain and a carboxy-terminal intracellular tail containing a conserved region called the Toll/interleukin-1 receptor (TIR) homology domain. The extracellular domain contains a varying number of LRR domains, which are presumably involved in ligand binding but may also be necessary for TLR dimerization.
PAMP ligands for most of the TLRs have been identified and include lipopolysaccharide (LPS) (recognized by TLR4), bacterial lipoproteins and lipoteichoic acid (recognized by TLR2), double stranded RNA (recognized by TLR3), flagellin (recognized by TLR5), single stranded viral RNA (recognized by TLR7 and TLR8), and viral and bacterial unmethylated CpG DNA (recognized by TLR9). In addition, host-derived ligands for several TLRs have been identified and include heat shock proteins (recognized by TLR4), chromatin-IgG complexes (recognized by TLR9), and endogenous mRNA (recognized by TLR3). No direct ligands have been identified for TLR 1 and TLR 6, but they appear to function as cofactors for TLR2.
Based on their ligands, TLRs 1, 2, 5 and 6 appear to specialize in recognition of products unique to bacteria and not made by the host. Their detection, therefore, affords a straight-forward self/non-self recognition. TLRs 3, 7, 8 and 9, on the other hand, specialize in viral detection and recognize nucleic acids, which are not unique to the microbial kingdom. In this case, self/non-self recognition is mediated not so much by the molecular nature of the ligands as by their accessibility to the TLRs. These TLRs are localized to intracellular compartments and detect viral nucleic acids in late endosomes-lysosomes. Because the host's nucleic acids are not normally accessible in these compartments, they do not trigger TLR-mediated signal transduction.
Once engaged, most TLRs initiate a signal transduction cascade leading to activation of NFκB via the adapter protein myeloid differentiation primary response gene 88 (MyD88) and recruitment of the IL-1 receptor associated kinase (IRAK). Phosphorylation of IRAK then leads to recruitment of TNF-receptor associated factor 6 (TRAF6), which then results in the phosphorylation of the NFκB inhibitor I-κB. As a result, NFκB enters the cell nucleus and initiates transcription of genes whose promoters contain NFκB binding sites, such as cytokines. TLR3, on the other hand, utilizes a MyD88-independent pathway to recruit TRIF to activate NFκB and IRF3, resulting in IFN-β production.
Various assays have been developed for identifying agonists and antagonists of TLRs (e.g., U.S. Patent Application publication Nos. 2004/0197865, 2004/0132079, 2004/022777, 2004/0014779, 2003/0166001, 2003/0104523, 2003/0044429, 2003/0022302). These assays, however, generally depend on the detection of delayed downstream events, such as cytokine production or reporter induction. Such delay in detection increases the likelihood that any toxicity associated with the test compound, the solvent, or the extracellular milieu will adversely affect the assay. As a result, there is an immediate need for a sensitive screening assay that allows detection of an immediate signal mediated by TLR engagement and materials for performing such an assay.